Sindbis virus, (SV) one of the simplest, enveloped RNA viruses, is the prototype virus of the family Togaviridae, genus alphavirus. It is transmitted to its vertebrate hosts by mosquitoes. Many viruses in the genus alphavirus are important causes of human disease, such as encephalitis. Several may also be considered possible bioterrorism agents. An essential step in the replication of Sindbis virus is the synthesis of a subgenomic (SG) RNA. This RNA serves as the message for the three structural proteins of the virus. The overall goal of this proposal is to study the means by which the synthesis of the SG RNA is regulated, and how the synthesis of the SG RNA is coordinated with synthesis of the genomic (G) RNA. Both of these plus-strand RNAs are made off the same minus strand RNA template. Virtually all studies of SG RNA synthesis to date have been done with whole cells, and have focused on definition of the SG promoter. We bring to our proposed studies, three new in vitro systems that we have developed, and a unique viral mutant, SV-pzf, that is deficient in the synthesis of SG RNA. The in vitro systems are 1) an assay for binding of the SV transcriptase to the SG promoter, 2) a cell-free system for making SG RNA which is dependent on addition of an exogenous promoter-template (PT) and 3) a cell-free system in which both SG and G RNA are made from the same minus-strand PT. These systems make possible completely new approaches for the study of SG RNA synthesis. Using these systems, we will examine not only the SG promoter, but also the role of nsP4, the viral RNA-dependent RNA polymerase, and the influence of NTP concentrations on the synthesis of SG RNA. Our first goal will be to reproduce in vitro the defect in SG RNA synthesis seen with SV-pzf. Our experiments will enable us to determine 1) the relative contributions of the SV-pzf mutations in nsP4, and in the SG promoter to the defect in SG RNA synthesis, 2) whether these mutations affect the binding of the transcriptase to the SG promoter. Our second goal is to characterize the 3'terminal sequence of the RNA (-) strand RNA in terms of the stem-loop structures needed for maximum activity of the G promoter, and to identify the amino acid sequence in nsP4 that binds to this promoter. Our third goal is to study the coordination of the synthesis of the SG and G RNAs, and to learn how the synthesis of these two RNAs is balanced in the infected cell. We will ask whether the SG and G promoters compete with each other in a cell-free system which makes both of these RNAs. We will also examine how the concentrations of the NTPs, which are the substrates of the replicase/transcriptase, influence the balance between synthesis of the SG and G RNAs. What we learn about the synthesis of SV SG RNA should have application to other viruses which make SG RNAs. As well, this new information could facilitate the development of SG RNA synthesis as a target for anti-viral therapy.